Type I interferon signaling induces a delayed antiproliferative response in respiratory epithelial cells during SARS-CoV-2 infection.

ABSTRACT: Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain unclear. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top four genes identified in our screen encode components of the same type I interferon (IFN-I) signaling complex­IFNAR1, IFNAR2, JAK1, and TYK2. The fifth gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response in both Calu-3 cells and iPSC-derived type 2 alveolar epithelial cells. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

Identification of cell type specific ACE2 modifiers by CRISPR screening.

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Human Norovirus Efficiently Replicates in Differentiated 3D-Human Intestinal Enteroids.

Human norovirus (HNoV) accounts for one-fifth of all acute viral gastroenteritis worldwide and an economic burden of ~$60 billion globally. The lack of treatment options against HNoV is in part due to the lack of cultivation systems. Recently, a model of infection in biopsy-derived human intestinal enteroids (HIE) has been described: 3D-HIE are first dispersed in 2D-monolayers and differentiated prior to infection, resulting in a labor-intensive, time-consuming procedure. Here, we present an alternative protocol for HNoV infection of 3D-HIE. We found that 3D-HIE differentiated as efficiently as 2D-monolayers. In addition, immunofluorescence-based quantification of UEA-1, a lectin that stains the villus brush border, revealed that ~80% of differentiated 3D-HIE spontaneously undergo polarity inversion, allowing for viral infection without the need for microinjection. Infection with HNoV GII.4-positive stool samples attained a fold-increase over inoculum of ~2 Log10 at 2 days postinfection or up to 3.5 Log10 when ruxolitinib, a JAK1/2-inhibitor, was added. Treatment of GII.4-infected 3D-HIE with the polymerase inhibitor 2'-C-Methylcytidine (2CMC) and other antivirals showed a reduction in viral infection, suggesting that 3D-HIE are an excellent platform to test anti-infectives. The transcriptional host response to HNoV was then investigated by RNA sequencing in infected versus uninfected 3D-HIE in the presence of ruxolitinib to focus on virus-associated signatures while limiting interferon-stimulated gene signatures. The analysis revealed upregulated hormone and neurotransmitter signal transduction pathways and downregulated glycolysis and hypoxia-response pathways upon HNoV infection. Overall, 3D-HIE have proven to be a highly robust model to study HNoV infection, screen antivirals, and to investigate the host response to HNoV infection. IMPORTANCE The human norovirus (HNoV) clinical and socio-economic impact calls for immediate action in the development of anti-infectives. Physiologically relevant in vitro models are hence needed to study HNoV biology, tropism, and mechanisms of viral-associated disease, and also as a platform to identify antiviral agents. Biopsy-derived human intestinal enteroids are a biomimetic of the intestinal epithelium and were recently described as a model that supports HNoV infection. However, the established protocol is time-consuming and labor-intensive. Therefore, we sought to develop a simplified and robust alternative model of infection in 3D enteroids that undergoes differentiation and spontaneous polarity inversion. Advantages of this model are the shorter experimental time, better infection yield, and spatial integrity of the intestinal epithelium. This model is potentially suitable for the study of other pathogens that infect intestinal cells from the apical surface but also for unraveling the interactions between intestinal epithelium and indigenous bacteria of the human microbiome.

Akt Plays Differential Roles during the Life Cycles of Acute and Persistent Murine Norovirus Strains in Macrophages.

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Host-Virus Chimeric Events in SARS-CoV-2-Infected Cells Are Infrequent and Artifactual.

The pathogenic mechanisms underlying severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection remain largely unelucidated. High-throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen- and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in transcriptome sequencing (RNA-seq) data from SARS-CoV-2-infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV-2 is a positive-sense RNA virus that replicates in the cytoplasm, it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events like mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with coronavirus disease 2019 (COVID-19) and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spiked-in RNA from an unrelated species, such as the fruit fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV-2 RNA-seq, we found that the frequency of HVC events was, in fact, not greater than this background "noise." Finally, we developed a novel experimental approach to enrich SARS-CoV-2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV-2-infected cells are extremely rare and are likely artifacts arising from random template switching of reverse transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV-2 fusion to cellular genes and/or integration into human genomes. IMPORTANCE The pathogenic mechanisms underlying SARS-CoV-2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known about the reasons some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV-2-infected cells and suggested that HVC events support potential "human genome invasion" and "integration" by SARS-CoV-2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here, we provide several lines of evidence suggesting that the observed HVC events are likely artifactual.

A novel druggable interprotomer pocket in the capsid of rhino- and enteroviruses.

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are "capsid binders" that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.

Severe Acute Respiratory Syndrome Coronavirus 2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 Replication in an Immunocompromised Patient.

We describe a case of chronic coronavirus disease 2019 (COVID-19) in a patient with lymphoma and associated B-cell immunodeficiency. Viral cultures and sequence analysis demonstrate ongoing replication of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for at least 119 days. The patient had 3 admissions related to COVID-19 over a 4-month period and was treated twice with remdesivir and convalescent plasma with resolution of symptoms. The patient's lack of seroconversion and prolonged course illustrate the importance of humoral immunity in resolving SARS-CoV-2 infection. This case highlights challenges in managing immunocompromised hosts, who may act as persistent shedders and sources of transmission.

Antiviral effects of bovine lactoferrin on human norovirus.

Human noroviruses cause significant morbidity and mortality worldwide, but lack approved antivirals or vaccines to treat or prevent infections. The recent development of two cell culture systems in human transformed B cells (BJABs) and non-transformed human intestinal enteroid cultures overcomes a main limitation in identifying molecules with anti-norovirus activities. Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals, with broad spectrum antimicrobial activities, including against the related murine norovirus in cell culture. In a Japanese clinical trial, ingestion of lactoferrin reduced the incidence of infectious gastroenteritis in the participants. Because human noroviruses were the most common cause of gastroenteritis in Japan during the clinical trial period, we sought to determine whether lactoferrin could inhibit infection with human norovirus. Our study, using a B cell culture model, demonstrates that lactoferrin reduces human norovirus infection. The mechanism of antiviral action is likely indirect and may involve the induction of innate interferon responses. Therefore, future studies are warranted to test the antiviral efficacy of lactoferrin against human norovirus infection in patients.

Viral engagement with host receptors blocked by a novel class of tryptophan dendrimers that targets the 5-fold-axis of the enterovirus-A71 capsid.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Differential antiviral activities of respiratory syncytial virus (RSV) inhibitors in human airway epithelium.

Objectives: We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. Methods: HuAECs were infected with RSV-A Long strain (0.01 CCID50/cell, where CCID50 represents 50% cell culture infectious dose in HEp2 cells) on the apical compartment of the culture. At the time of infection or at 1 or 3 days post-infection, selected inhibitors were added and refreshed daily on the basal compartment of the culture. Viral shedding was followed up by apical washes collected daily and quantifying viral RNA by RT-qPCR. Results: RSV-A replicates efficiently in HuAECs and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post-infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleoside) elicited a marked antiviral effect even when the start of treatment was delayed until 1 day or even 3 days after infection. Levels of the inflammation marker RANTES (mRNA) increased ∼200-fold in infected, untreated cultures (at 3 weeks post-infection), but levels were comparable to those of uninfected cultures in the presence of PC786, an RSV replication inhibitor, suggesting that an efficient antiviral treatment might inhibit virus-induced inflammation in this model. Conclusions: Overall, HuAECs offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds.

Fluorination of Naturally Occurring N6-Benzyladenosine Remarkably Increased Its Antiviral Activity and Selectivity.

Recently, we demonstrated that the natural cytokinin nucleosides N6-isopentenyladenosine (iPR) and N6-benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile of this class of compounds, we generated a series of fluorinated derivatives of BAPR and evaluated their activity on the replication of human enterovirus 71 in a cytopathic effect (CPE) reduction assay. The monofluorination of the BAPR-phenyl group changed the selectivity index (SI) slightly because of the concomitant high cell toxicity. Interestingly, the incorporation of a second fluorine atom resulted in a dramatic improvement of selectivity. Moreover, N6-trifluoromethylbenzyladenosines derivatives (9-11) exhibited also a very interesting profile, with low cytotoxicity observed. In particular, the analogue N6-(3-trifluoromethylbenzyl)-adenosine (10) with a four-fold gain in potency as compared to BAPR and the best SI in the class represents a promising candidate for further development.

The CREB3-Herp signalling module limits the cytosolic calcium concentration increase and apoptosis induced by poliovirus.

Poliovirus (PV)-induced apoptosis seems to play a major role in central nervous system (CNS) tissue injury, a crucial feature of the pathogenesis of poliomyelitis. We have previously shown that calcium (Ca2+) flux from the endoplasmic reticulum (ER) to the cytosol during PV infection is involved in apoptosis induction in human neuroblastoma cells. We show here that PV infection is associated with a transient upregulation of Herp (homocysteine-induced ER protein), a protein known to promote the degradation of ER-resident Ca2+ channels. Herp gene transcription is controlled by the transcription factor CREB3 (cAMP response element-binding protein 3). We found that the CREB3/Herp pathway limited the increase in cytosolic Ca2+ concentration and apoptosis early in PV infection. This may reduce the extent of PV-induced damage to the CNS during poliomyelitis.

The Golgi protein ACBD3, an interactor for poliovirus protein 3A, modulates poliovirus replication.

We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence.

Recrutement des kinases PI4KIII aux organelles de réplication virale au cours de l'infection par le poliovirus et d'autres virus à ARN de polarité positive.

One characteristic of infections with RNA viruses of positive polarity is the generation of new specialized membrane structures acting as platforms accommodating the complexes involved in replication of the viral genome. The functionality of these "replication organelles" is dependent on interactions between viral nonstructural proteins, recruited host factors and viral RNAs. Poliovirus, the causal agent of paralytic poliomyelitis, is the model most frequently used for identification of the viral and cellular components involved in this process. Several recent studies have suggested that the efficiency of genome replication for poliovirus and other members of the Picornaviridæ family results from the recruitment of a phosphatidylinositol (PI) kinase, PI4KIIIß (phosphatidylinositol-4-kinase IIIß), which generates a lipid membrane microenvironment rich in PI4P (phosphatidylinositol-4-phosphate) at sites of replication. The nonstructural protein 3A of these viruses has been shown to play a role in the enrichment of replication organelle membranes in PI4KIIIß, but the mechanisms of kinase recruitment seem to differ between members of this family of viruses. Hepatitis C, from the Flaviviridæ family, recruits another PI4KIII kinase, PI4KIIIα, to sites of replication, through another nonstructural protein, NS5A. In this review, we will describe the various recently proposed models and the potential role of PI4P lipids. Finally, we will show that PI4KIII kinases are potential targets for the development of antiviral drugs targeting many positive-polarity RNA viruses.

Bivalent norovirus mRNA vaccine elicits cellular and humoral responses protecting human enteroids from GII.4 infection.

Nucleoside-modified mRNA-LNP vaccines have revolutionized vaccine development against infectious pathogens due to their ability to elicit potent humoral and cellular immune responses. In this article, we present the results of the first norovirus vaccine candidate employing mRNA-LNP platform technology. The mRNA-LNP bivalent vaccine encoding the major capsid protein VP1 from GI.1 and GII.4 of human norovirus, generated high levels of neutralizing antibodies, robust cellular responses, and effectively protected human enteroids from infection by the most prevalent genotype (GII.4). These results serve as a proof of concept, demonstrating that a modified-nucleoside mRNA-LNP vaccine based on norovirus VP1 sequences can stimulate an immunogenic response in vivo and generates neutralizing antibodies capable of preventing viral infection in models of human gastrointestinal tract infection.

Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.